N-Glycosidase F Deglycosylation Kit
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1 For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. N-Glycosidase F Deglycosylation Kit Kit for the deglycosylation of asparagine-linked glycan chains on glycoproteins. Cat. No Kit for 12 deglycosylation reactions (including controls) Store at 15 to 25 C Instruction Manual Version 2, September 2003
2 1. Preface 1.1 Table of Contents 1. Preface Table of contents Kit contents Product overview Procedures and required materials Before you begin Protocol for complete deglycosylation Working procedure for estimating the number of glycan chains bound to a glycoprotein Interpretations of results Appendix References Related products
3 1.2 Kit contents Bottle Label Contents 1 N-Glycosidase F, recombinant Lyophilizate 150 U 2 Denaturation buffer, ph µl contains sodium phosphate and ionic detergent 3 Control glycoproteins human transferrin ribonuclease B, human α1-acid glycoprotein Lyophilized 150 µg of each protein 4 Reaction buffer, ph µl Contains sodium phosphate and nonionic detergent (the nonionic detergent is necessary to complex any excess ionic detergent used for denaturing) 5 Premixed protein molecular weight markers 10 µl (10 µg) of each protein Phosphorylase B, 97.4 kd Bovine serum albumin, 66.2 kd Aldolase, 39.2 kd Triose phosphate isomerase, 26.6 kd Trypsin inhibitor, 21.5 kd Lysozyme, 14.4 kd 3
4 2. Product overview Introduction For structural analysis of N (asparagine)-linked carbohydrate chains of glycoproteins, chemical hydrazinolysis and enzymatic methods are most widely employed to cleave all common classes of oligosaccharides. During hydrazinolysis, however the protein is destroyed and also degradation and modification of the released sugar chains have been observed (1,2). Enzymatic procedures are therefore the only methods allowing both the structural analysis of the glycan and the protein part. Several endoglycosidases useful for these applications have been described. Endoglycosidase H* and Endoglycosidase F1 for instance act only on high mannose type and hybrid type structures, while Endoglycosidase F2 prefers biantennary complex type chains. While these enzymes are useful tools for the selective removal and characterization of the individual glycan types (3), only N-glycosidase F is able to release all common classes of N-glycans from the protein backbone (4). N-glycosidase F is not a glycosidase but an amidase converting asparagine to aspartic acid in the following reaction: Protein-asn-GlcNAc-Glycan Protein-asp + NH 3 + GlcNAc-Glycan Not all carbohydrate chains on a glycoprotein are equally sensitive to N-glycosidase F. In many cases N-glycosidase F can cleave the intact glycoproteins (e.g., RNAse B, fetuin, human transferrin, ovomucoid and fibrinogen) (4,5). For other glycoproteins like for instance α 1 -acid glycoprotein the reducing of -S-S- bridges and denaturation of the protein using ionic detergents or chaotropic salts prior to the enzymatic treatment was found to be necessary (6,7). From a variety of denaturing conditions a procedure with optimum results for all glycoproteins has been developed as basis for this kit. It must be stated, however, that some modifications like α1,3-bound core fucose present on some plant and invertebrate glycoproteins allow only deglycosylation with N-glycosidase A* and not with N-glycosidase F (8). Reaction principle R 1 = Fucose -1,3 R 2 = Oligosaccharide 4
5 Application Purity Control glycoproteins Number of reactions Storage and stability The kit can be used to test for the existence of asparagine-linked glycan chains on glycoproteins. The kit also allows to estimate the number of glycan chains bound to a glycoprotein. After the deglycosylation reaction the protein can be analyzed on SDS-PAGE, where a shift to a lower apparent molecular weight after the reaction indicates the existence of asparagine linked glycan chains. The glycan chains can also be used for further structural and functional analysis. N-glycosidase F is produced as a recombinant protein from E. coli. It is highly purified and tested to be free of contaminating protease, exo- and endoglycosidase activities. Transferrin (from human serum): Transferrin has an apparent M r of 65 kd containing two glycan chains of sialylated bi- and triantennary complex type. The apparent M r is reduced to 60 kd upon digestion with N-glycosidase F (9). α 1 -acid glycoprotein (from human serum): α 1 -acid glycoprotein has an apparent M r of 45 kd containing five glycan chains of sialylated bi- tri- and tetraantennary complex type. The apparent M r is reduced to 22 kd upon digestion with N-glycosidase F (10). Ribonuclease B (from bovine pancreas): Ribonuclease B has an apparent M r of 17 kd containing one glycan chain of high mannose type. The apparent M r is reduced to 15 kd upon digestion with N-glycosidase F (11). The contents of the kit are sufficient for 10 deglycosylation reactions and two additional deglycosylations with the control glycoproteins. The kit is stable at -15 to -25 C until the expiration date printed on the label. The stability of the reconstituted solutions is indicated in chapter 3. Advantages Benefit fast easy versatile complete Feature The deglycosylation can be completed within 1 2 hours Only 3 easy working steps are necessary Unlike to chemical procedures, both, the glycan and the protein part can be analyzed after the reaction. All required buffers and reagents (except ß-mercaptoethanol) are supplied with the kit. 5
6 3. Procedures and required materials 3.1 Before you begin Additional equipment and reagents required Incubator for 37 C and 95 C equipment for SDS-PAGE and staining of gels lab centrifuge SDS-sample buffer: 125 mm Tris-HCl, ph 6.8, 10% glycerol (v/v), 2% SDS (w/v), 5% ß-mercaptoethanol, (v/v), 0.01% bromophenol blue (w/v). SDS-PAGE staining reagents Preparation of kit working solutions Please refer to the following table N-Glycosidase F Solution Composition/Preparation Storage/ Stability Reduced denaturation buffer Control glycoproteins Reaction buffer Premixed protein molecular weight markers Dissolve the lyophilizate in 125 µl of double dist. water. Thaw the frozen solution and warm to 37 C to dissolve all contents. Take the needed amount of denaturation buffer and add 1% ß-mercaptoethanol (v/v) before use. Dissolve the content in 50 µl of double dist. water. Thaw the frozen solution and warm up to 37 C to dissolve all contents. Dilute the molecular weight markers with 190 µl of SDS-sample buffer and heat for 3 min at 95 C immediately prior to use. Note: 5 µl of this solution is necessary for a cm minigel and staining with Coomassie. several months at 2-8 C up to 2 months at 15 to 25 C -15 to -25 C -15 to -25 C 6
7 3.2 Protocol for complete deglycosylation Before you begin Handling instructions Procedure For each sample to be analyzed label two eppendorf vials with e.g., + and -. In the same manner label two vials for the Control glycoproteins with C+ and C-. Usually the glycoprotein samples can be used without further treatment prior to digestion. If, however, the sample material contains ionic detergents, potassium ions or acidic buffers in high concentration a dialysis against double distilled water or neutral buffers is recommended. The denaturation buffer makes proteins very sensitive against proteolytic attack. Therefore, if a protease contamination of the sample can not be excluded, it is recommended to use Complete 1) Mini* (protease inhibitor cocktail tablets) for protease inhibition. The maximum amount of glycoprotein for 1 reaction is 100 µg. If higher amounts of glycoproteins are to be digested longer incubation times or multiple reactions are needed. Please refer to the following table. Step Action 1 Add 5 µl of your glycoprotein sample to the vials labeled with + and -. Add 5 µl of Control glycoprotein to vials the labeled with C+ and C-. 2 Add 5 µl of Reduced denaturation buffer to each vial. Incubate for 3 min at 95 C. Centrifuge down the contents after the heating step. 3 Add 10 µl of Reaction buffer to each vial and mix. 4 Add 10 µl of reconstituted N-glycosidase F to all vials labeled with + (including C+ ). Add 10 µl of Reaction buffer to all vials labeled with - (including C- ). Incubate for 1 hour at 37 C. Note: Both the Denaturation buffer and the Reaction buffer contain detergents which may interfere with further analysis. To remove these detergents add 1 ml of cold acetone to each vial, mix and centrifuge for 5 min at g. Then aspirate carefully 950 µl of the supernatant and reextract the pellet once again with acetone. Then dry the pellet using a vacuum concentrator. The dried pellet containing detergent free carbohydrates (together with proteins and buffer salts) can now be dissolved in water and further analyzed. 5 Mix an appropriate aliquot of the samples with an equal amount of SDSsample buffer and heat for 3 min to 95 C. Note: 10 µl of the sample solution is sufficient for a cm minigel and staining with Coomassie. 7
8 Example of the digested control glycoproteins An example of the digested control glycoproteins is shown in fig MW Transferrin Enzyme acid glycoprotein RNAse B Fig. 2: Lane 1, digested glycoproteins Lane 2, undigested glycoproteins Lane 3, protein molecular weight markers 3.3 Working procedure for estimating the number of glycan chains bound to a glycoprotein Handling instructions Before you begin Procedure Protocol 3.3 should only be performed, if protocol 3.2 worked properly and indicated the existence of N-glycosidic chains. Usually the glycoprotein samples to be digested can be used without further pretreatment. If, however, the sample material contains ionic detergents, potassium ions or acidic buffers in high concentration a dialysis against double distilled water or neutral buffers is recommended. For each sample to be analyzed label five eppendorf vials with 60, 45, 30, 15, and 0 to indicate the time (in minutes) of the N-glycosidase F reaction. Please refer to the following table. Step Action 1 Add 5 µl (with 5 50 µg) of glycoprotein sample to all vials. 2 Add 5 µl of Reduced denaturation buffer to each vial. Incubate for 3 min at 95 C. Centrifuge down the contents after the heating step. 3 Add 10 µl of Reaction buffer to each vial and mix. 4 Add an additional 10 µl of Reaction buffer to the vial labeled with 0 and mix. 5 Add 10 µl of N-glycosidase F to all vials except of the vial labeled with 0 and mix. Incubate all vials at 37 C. 6 At the time indicated on the vials add 30 µl of SDS-sample buffer and immediately heat for 3 min to 95 C. Then analyze on SDS-PAGE. 8
9 4. Interpretation of results Complete deglycosylation The following results can be obtained: Result Interpretation Recommendation The apparent molecular weights for the sample and the control glycoproteins decrease after N-glycosidase F treatment to one single band each upon SDS-PAGE. The apparent molecular weights for the control glycoproteins decrease after N- glycosidase F treatment to one band and the sample yields several bands upon SDS-PAGE. The apparent molecular weights for the sample and the control glycoproteins decrease after N-glycosidase F treatment to several bands each upon SDS-PAGE. N-glycosidase F treatment to one band and the sample does not show an increased mobility on SDS-PAGE. The sample is a glycoprotein containing one or more asparagine bound glycosidic chains. The sample is a glycoprotein containing more than one asparagine bound glycosidic chain but, due to partial resistance to N-glycosidase F, is not cleaved completely. The sample is not denatured properly or it contains inhibiting agents to N-glycosidase F. The sample is heterogeneous because of reasons other than glycosylation. Incomplete denaturation of proteins or the activity of N- glycosidase has decreased. The sample does not contain asparagine bound glycan chains. The sample contains inhibiting agents to N-glycosidase F. To estimate the number of glycosidic chains present on the sample glycoprotein perform protocol 3.3. Add a second aliquot of N- glycosidase F after 1 hour. The presence of inhibitors can be assessed by coincubating the control glycoproteins with the sample. Inhibitors may be removed by dialyzing the sample. Increase the denaturation time to 10 min. If the result is the same use a higher amount or a fresh vial of N-glycosidase. The absence of glycosylation may be proved by a glycoprotein specific staining using our DIG Glycan Detection Kit*. The presence of inhibitors can be assessed by coincubating the control glycoproteins with the sample. Inhibitors may be removed by dialyzing the sample. 9
10 Estimation of the number of glycan chains (3.3) Protocol 3.3 should only be performed if protocol 3.2 worked properly and indicated the existence of N-glycosidic chains. The following results can be obtained: Result Interpretation Recommendation Even at the shortest incubation time only one band at a lower apparent molecular weight appeared. More than one band (= n) appear at a lower molecular weight. The lowest band becomes more intensive with incubation time, while the band of the original undigested sample disappears There is only one glycan chain present. N-glycosidase F removed several chains, which are very sensitive to N-glycosidase. The sample contains N- asparagine glycan chains. Repeating the experiment using 6 twofold dilutions of the enzyme starting with 50 U/ml and a constant incubation time of 15 min. 5. Appendix 5.1 References 1 Takasaki, S. et al (1980) Meth. Enzymol. 83, Egge, H. et al (1983) FEBS Lett. 156, Plummer, T.H. & Tarentino A.L. (1991) Glycobiology 1, Tarentino, A.L. & Plummer, T.H. (1987) Meth. Enzymol. 138, Langer, B.G. et al (1987) Anal Biochem. 166, Nuck, R. et al (1990) Glycoconj. J. 7, Haselbeck, A. & Hösel, W. (1988) Topics in Biochem. 8, 1. 8 Tretter, V. et al (1991) Eur. J. Biochem. 199, Spik, G. et al. (1975) FEBS Lett. 50, Treuheit, M.J. et al (1992) Biochem. J. 283, Laing, C.J. et al (1980) J. Biochem. 88,
11 5.2 Related products For further information contact our Complete site for Protease inhibition at A Protease inhibitor guide is available at Product Pack size Cat. No. Inhibition of proteases Complete, Mini 25 tablets à 10 ml Complete Protease Inhibitor Cocktail Tablets Complete EDTA free Inhibitor Cocktail Tablets Complete Mini EDTA free Inhibitor Cocktail Tablets 20 tablets 3 x 20 tablets tablets tablets Protease Inhibitor Set 1 set Detection of carbohydrates in glycoconjugates DIG Glycan Detection Kit Kit for 100 reactions Deglycosylation of high mannose type glycans Endoglycosidase H 1 U (200 µl) 2.5 U (500 µl) Deglycosylation of O-linked carbohydrates O-Glycosidase 25 mu Deglycosylation of N-linked carbohydrates with α1,3 bound core fucose N-Glycosidase A 5 mu * available from 1) Complete is a trademark of a Member of the Roche Group. 11
12 to order, solve technical queries, find product information, or contact your local sales representative. Please visit our new Online Technical Support Site Roche Diagnostics GmbH Nonnenwald Penzberg Germany
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